In a study published in Nucleic Acids Res, a research team led by Prof. ZHOU Xiao-Long from the Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences demonstrated the first in vitro activity reconstitution of eukaryotic tRNA ac4C12 modification and proposed the principles underlying tRNA substrate selection strategy.
RNA acetylation is a prevalent post-transcriptional modification that occurs in a wide variety of RNAs, including messenger RNA (mRNA), transfer RNA (tRNA) and ribosomal RNA (rRNA). Currently, two main types of tRNA acetylation modifications have been identified, namely N6-acetyladenosine (ac6A) and N4-acetylcytidine (ac4C). While ac6A is found in archaeal tRNAs only, ac4C is present in all domains of life.
In eukaryotic cytoplasmic tRNAs, ac4C is found exclusively at the middle position of the 11CCG13 motif (position 12, ac4C12) in the D-arm of class II tRNASer and tRNALeu with long variable arms. Recently, the enzyme responsible for ac4C12 biogenesis was identified to be the RNA acetyltransferase Kre33/NAT10, which needs to work alongside Tan1/THUMPD1 in both yeast and human cells. However, modification activity of the eukaryotic tRNA ac4C12 has never been successfully reconstituted in vitro for nearly six decades, which has greatly impeded the understanding of the molecular mechanism and putative role of ac4C12 in tRNA structure and function.
In this work, the researchers purified a truncated form of Kre33 and full-length Tan1. Using acetyl-CoA as an acetylation donor, they successfully reconstituted ac4C12 modification of both tRNASer and tRNALeu. They elucidated the tRNA recognition mechanism involved in ac4C12 biogenesis and proposed a molecular ruler mechanism for substrate selection by Tan1 as a distance-measuring strategy. They were also able to modify various non-substrate cytoplasmic tRNAs by introducing a single A13G mutation. Moreover, they realized site-specific ac4C modifications in small RNAs depending on the tRNA recognition mechanism by Kre33–Tan1. In vitro and in vivo data suggested that tRNA recognition by Tan1 was facilitated by coordinated protein–RNA interactions. They determined that ac4C12 alone had no effect on the Tm values of tRNA transcripts, at least in vitro, or on the charging levels of tRNASer and tRNALeu in vitro and in vivo.
Taken together, this comprehensive study reveals the mechanism of substrate-specific recognition by the acetylation-modification complex Kre33-Tan1, and achieves targeted and efficient acetylation modification on tRNA and small RNA molecules. This study provides a solid basis for further research into the molecular mechanism, structural basis and functional significance of eukaryotic ac4C12 modification, as well as the molecular etiology of ac4C12-associated diseases.
Contact: xlzhou@sibcb.ac.cn
Reference: https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gkae262/7645239?login=true
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